Subscriber access provided by UNIV OF NEW ENGLAND ARMIDALE
Article
Metabolomics reveal optimal grain preprocessing (milling) toward rice koji fermentation Sunmin Lee, Da Eun Lee, Digar Singh, and Choong Hwan Lee J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b05131 • Publication Date (Web): 02 Mar 2018 Downloaded from http://pubs.acs.org on March 3, 2018
Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
Journal of Agricultural and Food Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.
Page 1 of 33
Journal of Agricultural and Food Chemistry
Metabolomics reveal optimal grain pre-processing (milling) toward rice koji fermentation Sunmin Lee, Da Eun Lee, Digar Singh, Choong Hwan Lee* Department of Bioscience and Biotechnology, Konkuk University, Seoul 05029, Republic of Korea;
[email protected] (S.L.);
[email protected] (D.E.L);
[email protected] (D.S.);
[email protected] (C.H.L)
*Corresponding author Choong Hwan Lee Department of Bioscience and Biotechnology, Konkuk University, Seoul 05029, Republic of Korea Tel.: +82-2-2049-6177; Fax: +82-2-455-4291; E-mail address:
[email protected] 1
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
1
ABSTRACT
2
A time-correlated mass spectrometry (MS) based metabolic profiling was performed for
3
rice koji made using the substrates with varying degrees of milling (DOM). Overall, 67
4
primary and secondary metabolites were observed as significantly discriminant among
5
different samples. Notably, a higher abundance of carbohydrate (sugars, sugar alcohols,
6
organic acids, phenolic acids) and lipid (fatty acids, lysophospholipids) derived metabolites
7
with enhanced hydrolytic enzyme activities were observed for koji made with substrate's
8
DOM 5–7, at 36 h. The antioxidant secondary metabolites (flavonoids and phenolic acid)
9
were relatively higher in koji with substrate's DOM 0, followed by DOM 5 > 7 > 9 and 11, at
10
96 h. Hence, we conjecture that the rice substrate pre-processing between DOM 5-7 was
11
potentially optimal toward koji fermentation with end-product being rich in distinctive
12
organoleptic, nutritional, and functional metabolites. The study rationalizes the substrate pre-
13
processing steps vital for commercial koji making.
14
KEYWORDS: Rice Koji, Degree of milling, Fermentation, Mass spectrometry, Enzyme
15
activity
2
ACS Paragon Plus Environment
Page 2 of 33
Page 3 of 33
Journal of Agricultural and Food Chemistry
16
1. INTRODUCTION
17
Fermentation, a customary food processing and preserving method, can be traced back to
18
thousands of years in pre-historic era.1 Considering the modern perspectives, the fermented
19
food products are globally relished as functional foods with the capacity to function as health
20
aids.2 Typically, a food fermentative bioprocess is carried out using diverse microflora
21
including fungi, yeast, and bacteria to improve the nutritional value and food safety with
22
enhanced organoleptic properties.3,4
23
The koji, being an inextricable component or starter for numerous fermented foods,
24
condiments, and beverages typical to East Asia, employed largely in food fermentative
25
bioprocesses.5 Koji is made through solid-state fermentation by inoculating steamed rice
26
grains with molds (A. oryzae), which germinates to secrete hydrolytic enzymes triggering
27
fermentation.6 Since nearly two millennia, Aspergillus strains have been used for the koji
28
fermentation, employed typically for rice wines (makgeoli, sake), soy sauce (ganjang, shoyu),
29
fermented soybean (doenjang, miso), soy-pepper paste (gochujang), and distilled spirits
30
(shochu).7 Particularly, rice koji fermentation with Aspergillus oryzae primarily occurs
31
through carbohydrate metabolism by secreted hydrolytic enzymes.8 In particular, the
32
hydrolytic enzymes secreted by A. oryzae (koji mold), modulates substrate textures through
33
affecting the release of metabolic gamut such as mono-sugars, amino acids, and fatty acids.9
34
In the fermentation process of any product, the starting substrate and inoculum are critical
35
to the quality of fermentation end-products. Previously, we correlated structural contours and
36
metabolic compositions of different rice varieties with culture growth, secreted enzymes as
37
well as metabolites in koji.10 Yoshizaki et al. have also reported the effects of three types of
38
rice koji (yellow, white, and black) on the volatile aroma compounds using gas
39
chromatography-mass spectrometry (GC-MS) based profiling.11 Functionally, the rice is a 3
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
40
valuable and rich source of antioxidants including phenolic compounds and has a hard husk
41
(hull) that protects the endosperm. Most of these compounds are distributed in different
42
portions of rice grain, particularly accessible through milling process. The commercial rice-
43
milling process generates products like husk and bran (pericarp) with low-value fractions.
44
After the husk is removed, the remainder is known as brown rice, which includes the bran,
45
embryo, and endosperm.12 Brown rice retains its bran layer and embryo bud, while the bran
46
and embryo of white rice are removed during complete milling. Liu et al. have highlighted
47
the effects of varying degrees of milling (DOM) on total flavonoid and phenolic
48
compositions as well as the associated antioxidant activities for brown rice .13 Determining an
49
appropriate DOM is vital for improving nutrient utilization and controlling potential hazards
50
associated with anti-nutritional factors to avoid nutrient loss due to over-milling.14
51
Metabolomics have increasingly been used to study the nutritional, functional, and
52
organoleptic aspects of fermented foods in a high-throughput manner owing to the
53
technological and computational advancements in mass spectrometry (MS) and related
54
databases, respectively. In recent years, the metabolomic perspectives of numerous fermented
55
foods viz., koji, doenjang, and gochujang, have been comprehensively studied to gain insights
56
of the corresponding nutritional, functional, and quality biomarkers.10,15,16 Herein, we
57
examined the subtle effects of rice substrate milling on metabolomic profiles and associated
58
biochemical phenotypes viz., enzyme activities and antioxidant phenotypes.
59 60
2. MATERIALS AND METHODS
61
2.1. Chemicals and Reagents. HPLC grade water, methanol, and acetonitrile were obtained
62
from Thermo Fisher Scientific (Waltham, MA, USA). Analytical grade chemicals viz., acetic
63
acid, 2,2′-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), casein 4
ACS Paragon Plus Environment
Page 4 of 33
Page 5 of 33
Journal of Agricultural and Food Chemistry
64
from bovine milk powder, Folin-Ciocalteu’s phenol, formaldehyde solution, formic acid,
65
methoxyamine hydrochloride, p-nitrophenol, p-nitrophenol β-D-glucopyranoside (p-NPG),
66
potassium persulfate, pyridine, sodium hydroxide, sodium acetate, starch, N-methyl-N-
67
(trimethylsilyl) trifluoroacetamide, 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid
68
(Trolox), and tyrosine were purchased from Sigma-Aldrich (St. Louis, MO, USA). Sodium
69
carbonate, sodium dihydrogen phosphate, and disodium hydrogen phosphate were purchased
70
from Junsei Chemical Co., Ltd. (Tokyo, Japan). Trichloroacetic acid was obtained from
71
Merck Millipore Co. (Darmstadt, Germany).
72
2.2. Rice Koji fermentation with substrates having varying DOM and sample harvest.
73
Brown rice samples of a Korean cultivar, Jinsang, were used in this study towards the
74
fermentative production of rice koji samples and differentially milled using a polishing
75
machine (Model MP-220, Yamamoto Co., Tokyo, Japan) to obtain rice samples with different
76
DOM. Each DOM was represented in terms of the proportion of embryo bud and bran layer,
77
remained in rice (Table 1). The 5 milled rice categories viz., DOM 0, DOM 5, DOM 7, DOM
78
9, DOM 11, were collected and stored at -20°C. The koji mold Aspergillus oryzae (KCCM
79
12698) procured from the Korean Culture Center of Microorganisms (KCCM, Seoul, Korea),
80
was used as culture inoculum. A. oryzae was maintained on Malt Extract Agar (malt extract:
81
20 g, glucose: 20 g, peptone: 1 g, and agar: 20 g per liter) at 28°C. The fermentative
82
bioprocess steps of koji production were adapted from Lee et al.10 The fermented rice koji
83
samples were harvested at every 12 h (up to 96 h) and stored at deep freezing conditions (-
84
80°C) until analyses.
85
2.3. GC-TOF-MS Analysis. All sample extraction and derivatization steps were performed
86
as described by Lee at al.10 Gas chromatography time-of-flight MS (GC-TOF-MS) analysis
87
was performed on an Agilent 7890A GC system (Santa Clara, CA, USA) with a Pegasus HT 5
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
88
TOF-MS (Leco Corporation, St. Joseph, MI, USA). An RTx-5MS (30 m length × 0.25 mm
89
i.d., J & W Scientific, Folsom, CA, USA) was used a carrier gas (helium) at a constant flow
90
rate of 1.5 mL/min. The injector and ion source temperature were maintained at 250°C and
91
230°C, respectively. The oven temperature was maintained at 75°C for 2 min, and then
92
increased to 300°C at 15°C/min, which was sustained for 3 min. Next, 1 µL of sample was
93
injected with a mass scan range of 50–800 m/z. Overall, three samples as well as analytical
94
replicates were maintained for each variant.
95
2.4. LC-ESI-MS Analysis. The samples were extracted and analyzed for secondary
96
metabolites using ultra-high-performance liquid chromatography linear trap quadrupole ion
97
trap tandem mass spectrometry (UHPLC-LTQ-IT-MS/MS) and ultra-performance liquid
98
chromatography quadruple time-of-flight mass spectrometry (UPLC-Q-TOF-MS), using the
99
protocols described by Lee at al.10 Samples were separated on a syncronis C18 column with
100
100 mm × 2.1 mm, 1.7 µm particle size (Thermo Scientific). The mass spectra and
101
photodiode array range in both positive and negative ion mode were tuned for 100–1000 m/z
102
and 200-600 nm, respectively.
103
2.5. Data Processing and Multivariate Statistical Analysis. The raw datasets from GC-
104
TOF-MS and UHPLC-LTQ-IT-MS/MS analyses were transformed to netCDF (*.cdf) format
105
using Leco ChromaTOF and Thermo Xcalibur software, respectively. The respective netCDF
106
(*.cdf) files were subjected to MetAlign software (http://www.metalign.nl) mediated data
107
processing as described previously by Lee et al.10,
108
contained the suitable peak mass (m/z), retention times (min), and peak area information as
109
variables, was evaluated using SIMCA-P+ 12.0 software (Umetrics, Umea, Sweden) for
110
multivariate statistical (MVS) analysis. The data sets were log-transformed and unit variance
111
was scaled prior to principal component analysis (PCA) and partial least-squares discriminant
15
The resulting data matrix, which
6
ACS Paragon Plus Environment
Page 6 of 33
Page 7 of 33
Journal of Agricultural and Food Chemistry
112
analysis (PLS-DA) to compare the five rice koji. Significant differences (p-value < 0.05)
113
were tested by one-way analysis of variance using PASW Statistics 18 (SPSS, Inc., Chicago,
114
IL, USA). The putative metabolite identification was performed through matching respective
115
molecular weights and formula, retention time, mass fragmentation patterns, and UV
116
absorbance data available in literature and our in-house library (Table S1 and S2).
117
2.6. Enzyme Activities. The enzymatic activity assays including amylase, protease, β-
118
glucosidase were conducted according to the protocols described by Lee et al.10 Each rice koji
119
sample (10 g) in 90 mL of water was extracted by shaking in an incubator at 120 rpm and
120
30°C for 1 h. To evaluate enzymatic activities, filtered supernatants were used.
121
2.7. Determination of Antioxidant Activity. To determine the antioxidant activity of rice
122
koji samples, ABTS and FRAP assays were performed in triplicate. The ABTS antioxidant
123
assay was performed using the method partially adapted from Re et al. and Lee et al.10, 17 For
124
analysis, the ABTS stock solution was diluted using distilled water achieving a solution with
125
absorbance of 0.7 ± 0.02 at 750 nm. Each sample extract (20 µL) was incubated with ABTS
126
solution (180 µL) in 96-well plate, and the reaction was incubated at room temperature for 6
127
min under dark, and the absorbance was measured at 750 nm. The FRAP assay was
128
performed using a mixture of 300 mM acetate buffer (pH 3.6), 20 mM iron(III) chloride, and
129
10 mM TPTZ solution in 40 mM HCl (10:1:1, v:v:v). For analysis, 10 µL of sample was
130
added 300 µL of FRAP reagent and incubated at room temperature for 6 min. Absorbance
131
was measured at 570 nm. The results are represented as the Trolox equivalent antioxidant
132
capacity concentration (mM) per milligram of koji. The standard curves of Trolox ranged
133
from 0.0156 to 0.5 mM.
134
2.8. Physicochemical Characteristics. For evaluating physicochemical characteristics, 3 g
135
of koji samples were extracted with 30 mL of distilled water at 30°C for 1 h and 120 rpm. 7
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
136
The content of total sugar contents at 25°C expressed as °Brix was determined using a digital
137
refractometer (HI 96811, Hannah Instruments, Woonsocket, RI, USA). pH was measured
138
using a pH meter (Thermo). Titratable acidity was measured by titrating the samples with 0.1
139
N NaOH solution to a pH value of 8.4. The amino-type nitrogen contents were estimated by
140
adding 36% formaldehyde solution (20 mL) to the titrated samples, followed by sample re-
141
titration to pH 8.4 using 0.1 N NaOH solution. Amino-type nitrogen contents were
142
quantitatively estimated based on the titrated volume of 0.1 N NaOH and transformed to total
143
amino acid content.
144
3. RESULTS
145
3.1. Metabolic Profiling and Multivariate Analyses for Rice Koji fermented using the
146
Substrates with Varying DOMs. Differential metabolomes among the rice koji samples
147
made using the substrates with varying DOM were examined using multivariate analyses i.e.,
148
PCA and PLS-DA, based on GC-MS and LC-MS datasets. The PCA and PLS-DA model
149
based on rice koji samples according to the DOM are shown in Figure 1 and Supplementary
150
Figure 1, respectively. The principal component analysis (PCA) is an unsupervised,
151
multivariate statistical method for transforming an original set of correlated variables to a
152
new set of uncorrelated variables, called principal components (PC). On the other hand,
153
partial least squares discriminant analysis (PLS-DA) is commonly applied to evaluate the
154
clear distinctions between groups (variables) within the observed datasets.
155
The PCA score plot derived from GC-TOF-MS (Figure 1A) and UHPLC-LTQ-ESI-IT-
156
MS/MS (Figure B) data sets showed 51.2% (PC1: 39.8% and PC2: 11.4%) and 21.7% (PC1:
157
18.6% and PC2: 3.1%) variances, respectively. The PLS-DA model showed a similar pattern
158
of metabolic profiles compared to the PCA model indicating a steady alteration in rice koji
159
metabolomes during the course of fermentation. Intriguingly, each rice koji with different 8
ACS Paragon Plus Environment
Page 8 of 33
Page 9 of 33
Journal of Agricultural and Food Chemistry
160
DOM substrates exhibited different metabolite changes. However, similar patterns were
161
observed between rice koji with substrates having DOM 5 and 7 as well as DOM 9 and 11
162
samples.
163
Differential variables were selected based on variable importance in projection (VIP >1.0)
164
values and p-values (p < 0.05) obtained by PLS-DA model. The variable importance in
165
projection (VIP) values reflects the statistical significance of each variable in the model. A
166
total of 54 primary metabolites by GC-TOF-MS data (18 amino acids, 7 fatty acids, 11
167
organic acids, 15 sugars and sugar alcohols, and 4 others) were identified using standard
168
compounds, mass fragment patterns, of NIST and an in-house library (Table S1). Further, 13
169
secondary metabolites by UHPLC-ESI-LTQ-MS/MS data were selected as major compounds
170
related to the discrimination between rice koji with different DOM (Table S2). In particular,
171
apigenin-6-C-glucosyl-8-C-arabinoside, isovitexin-2''-O-glucoside, tricin-O-rutinoside, tricin,
172
pinellic acid, and 8 lysophospholipids including lysophosphatidylcholines (lysoPC) and
173
lysophosphatidylethanolamine (lysoPE) were identified by comparing the data to an in-house
174
library and published literature.
175
3.1.1 Temporal Primary Metabolomes for Rice Koji fermented using Substrates with
176
Varying DOMs. To visualize discriminative primary metabolites according to fermentation
177
time, a heat-map was made based on the GC-TOF-MS data. The trends of variations in
178
metabolite levels according to fermentation time are shown in Figure 2, with their relative
179
levels are represented as fold-changes normalized to the respective value of unfermented rice
180
with different DOM. The trends of sugar and sugar alcohol derivatives viz., adonitol, dulcitol,
181
gluconate, glycerate, maltose, myo-inositol, raffinose, sucrose, sorbitol, and xylose, were
182
gradually increased. The relative abundance of glucose was increased until 24 h and then
183
decreased until 96 h. In amino acid metabolism, the relative levels of serine, glycine, 9
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
184
isoleucine, threonine, valine, methionine, aspartate, alanine, phenylalanine, tryptophan, lysine,
185
gaba, ornithine, and pyroglutamate were increased, whereas those of asparagine and
186
glutamate were gradually decreased. Among the fatty acids, particularly palmitic acid,
187
linoleic acid, elaidic acid, oleic acid, linolenic acid, stearic acid, and myristic acid, were
188
increased. Metabolites related to the tricarboxylic acid (TCA) cycle such as citrate, malate,
189
fumarate, and succinate increased with fermentation time.
190
3.1.2 Relative Disparity in the Levels of Discriminant Metabolites in Rice Koji made
191
using the Substrates with Varying DOMs. To illuminate the relative levels of discriminant
192
metabolites among rice koji types made using the substrate with varying DOM representing
193
each fermentation time point, a heatmap was used for indicating the highest levels of
194
respective metabolites (Table 2). The comparison with substrates (unfermented rice) revealed
195
that most metabolites such as amino acids, organic acids, sugars and sugar alcohols and
196
secondary metabolites were relatively higher in DOM 0 koji, whereas fatty acids were highest
197
in DOM 5 koji. Depending upon the fermentation time point, the levels of metabolites in
198
various rice koji types changed from DOM 0 to DOM 5–7 at 24 h, and subsequently from
199
DOM 5–7 to DOM 7–9 at 36 h. Between 48–72 h, these metabolic trends changed in the rice
200
koji made with substrates having DOM 7–11. In contrast, the highest levels of amino acids,
201
sugars and sugar alcohols, nucleotide, fatty acids, and organic acids except secondary
202
metabolites were observed in DOM 0 rice koji samples at 96 h.
203
3.2 Variations in Enzymatic Production, Bioactivities, and Physiological Characteristics
204
for Rice Koji Types made using the Substrates with Varying DOM. The fermentation time
205
correlated enzymatic activities for different rice koji types made using the substrates with
206
varying DOM were determined to estimate the differential secretion of hydrolytic enzymes
207
by A. oryzae (Figure 3 A–C). In general, the amylase, protease, and β-glucosidase activities 10
ACS Paragon Plus Environment
Page 10 of 33
Page 11 of 33
Journal of Agricultural and Food Chemistry
208
were increased linearly with fermentation time in different rice koji types. The protease and
209
β-glucosidase activities were highest in the koji samples with DOM 7 substrates, whereas
210
amylase activity showed higher values in koji samples with DOM 0–9 substrates, except for
211
samples with DOM 11. Interestingly, amylase activity for all koji types regardless of DOM
212
was highest at 24 h, while protease and β-glucosidase activities increased linearly until 96 h.
213
Measurement of the functional phenotypes, i.e., ABTS and FRAP, to determine antioxidant
214
activities showed that the rice koji samples with DOM 0 substrates had the highest
215
antioxidants levels followed by DOM 5 > DOM 7 >DOM 9, 11 substrates made rice koji
216
(Figure 3 D and E). Hence, we assume that the antioxidant activities of koji increased linearly
217
until 96 h, regardless of DOM of the substrates.
218
4. DISCUSSION
219
We employed the metabolomic approaches to evaluate the comprehensive metabolic and
220
biochemical events underlying fermentative koji preparation with rice substrates having
221
varying DOM. We observed that the differential metabolomes of rice koji types were the
222
direct biochemical functions of (1) time-correlated metabolism, and (2) enzyme activities, by
223
koji mold subjected to fermentation on rice substrates with varying DOM. During the course
224
of fermentative growth, the spore count for A. oryzae was steadily increased while the
225
moisture content was decreased. There were no significant differences observed for mold
226
growth and sporulation among the koji samples with 5 different DOMs. We assumed that the
227
decrease of moisture content in koji was probably due to consumption of water by Aspergillus
228
growth as well as through evaporation.18
229
The multivariate analysis for various rice koji extracts based on GC-TOF-MS datasets
230
indicated a fermentation time-correlated metabolic profile for primary metabolites regardless
231
of the DOM (Figure 1A). On the other hand, the multivariate analysis based on UHPLC11
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
232
LTQ-ESI-IT-MS/MS datasets showed a secondary metabolite profile depending on various
233
DOM of rice substrates (Figure 1B). As shown in Figure 2, the primary metabolites altered
234
briskly with enzyme activities over time i.e., increased abundance of metabolites related to
235
sugar metabolism with increase in amylase and β-glucosidase activities at 24 h and 72 h,
236
respectively. Similarly, the gradual changes in amino acid levels were recorded with
237
enhanced protease activity. The fatty acid levels were also increased at 24 h.
238
4.1. Time Correlated Metabolic Alterations for Rice Koji Independent of Substrate's
239
DOM. The organic acids produced in the TCA cycle viz., citrate, fumarate, malate, and
240
succinate acts as intermediates to various metabolic pathways (Figure 2). With an increase in
241
organic acids contents, the pH decreased with an accompanying increase in titratable acidity
242
of the rice koji samples (Figure 4C and D). The production of citric acid used in the dairy,
243
food, beverage, pharmaceutical, and biochemical industries is extensively carried out using
244
Aspergillus species.19 The fumaric acid is used as a starting metabolite for the synthesis of
245
polymers, while the malate and succinate has wide applications in the food, beverage, and
246
pharmaceuticals industries.20
247
Among the sugars, the glucose levels showed the highest levels at 24 h unlike other sugar
248
metabolites, followed by its steep decline levels during the later stages of koji fermentation.
249
In particular, glucose is a major carbon source hydrolyzed from the substrate starch by fungal
250
enzymes including amylase and β-glucosidases. The sugars are further metabolized to sugar
251
alcohols through alcoholic fermentation.21 The sugar alcohols are produced during microbial
252
fermentation through multiple fermentation pathways.22 For example, erythritol, a four-
253
carbon sugar alcohol that is widely distributed in nature such as in foods, is produced
254
industrially via the pentose phosphate pathway beginning with enzymatic hydrolysis of the
255
starch from rice to generate glucose. Similarly, Sorbitol can be obtained by simultaneous 12
ACS Paragon Plus Environment
Page 12 of 33
Page 13 of 33
256
Journal of Agricultural and Food Chemistry
hydrolysis and reduction of glucose to change the aldehyde group to a hydroxyl groups.
257
The proteolytic enzymes produced by Aspergillus species release the organic nitrogen
258
contents from complex proteins to be used as a nitrogen source essential for growth and
259
metabolism.23,24 The rice-koji fermentation with Aspergillus spp. effectively increased the
260
contents of free amino acids (alanine, glycine, and serine) related to sweetness and nutritional
261
properties.25 The total amino acid contents including those of aromatic amino acids
262
(phenylalanine and tryptophan) and branched-chain amino acids (leucine, isoleucine, and
263
valine) were increased with enhanced protease activities over time (Figure 2, 3B, & 4F).
264
Functionally, the biosynthesis of aromatic amino acid in A. fumigatus has been linked with its
265
antifungal properties, while the branched-chain amino acids in A. nidulans are reportedly
266
vital for building proteins.26,27 In our study, we observed a decrease in glutamate contents
267
coupled with an increase in other amino acids contents, especially those of γ–aminobutyric
268
acid (GABA) over time. It has earlier been reported that, A. oryzae contains a gene for
269
glutamate decarboxylase (GAD, E.C. 4.1.1.15) which produces GABA via decarboxylation of
270
glutamic acid during cultivation of the spore suspension.28 GABA was employed in
271
functional foods for reducing blood pressure, promoting better sleep, and diuretic effects.29-31
272
The relative levels of unsaturated fatty acids, saturated fatty acids, and lysophospholipids
273
were increased with the biosynthesis as well as bioconversion of fatty acids (Figure 2).
274
Whole rice bran lipids induce the lipid metabolism. This means that fungi generate excess
275
lipids by fermenting the substrate materials as well as synthesize their own lipids for fungal
276
biomass production.32 Earlier, Abu et al. have reported that solid-state fermentation with A.
277
oryzae typically increases the concentrations of lipids, primarily including C16:0, C18:0, and
278
C18:1.33
279
4.2. Metabolic Alterations for Rice Koji Depending on Substrate's DOM. A rice grain has 13
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
280
different biomolecular compositions for its different parts arranged from surface to inside
281
including outer husk or hull, bran, embryo, and innermost endosperm. A variety of
282
metabolites in rice viz., phenolic acids, cinnamic acids, anthocyanins, flavonoids, steroidal
283
compounds, polymeric carbohydrate, and proteins are nutritionally vital as health-promoting
284
functional foods.34 Following the milling process, rice contains less of the embryo bud and
285
bran layer (Table 1). We detected higher proportions of amino acids, organic acids, sugars and
286
sugar alcohols, and lysophospholipids together contributing to the high antioxidant activities
287
for DOM 0 rice koji as compared to those for DOM 5–11 koji samples (Table 2, Figure 3 D
288
and E), maintaining similar pH and titratable acidity (Figure 4 C–D). Though, the relative
289
abundance for most metabolites in rice koji, made using the substrates with intermediate
290
DOM, were increased during the course of fermentation, the metabolite abundance for
291
unfermented DOM 0 substrate was comparatively higher than substrates with increased DOM
292
(Table 2).
293
Given the proportion of the bran layer in rice after different DOM, rice koji was relatively
294
affected by various microbial enzymes and associated bioactivities. The protease and β-
295
glucosidase activities were observed varying in the order of substrate's DOM 5 > 7 > 0 > 9 >
296
11, while the amylase activity was observed unaffected by the DOM. Accordingly, amino
297
acids, sugars, and sugar metabolism were highest in DOM 5-7 sample during the middle of
298
fermentation at 24–72 h. Intriguingly, most of the primary metabolites were highest in the
299
koji samples with DOM 0, at the end 96 h fermentation. Mechanistically, β-Glucosidase
300
cleaves glucosidic linkages in polysaccharides and flavonoid glycosides and hydrolyzes them
301
into oligo- or mono-saccharides and corresponding flavonoid aglycones to improve
302
enzymatic saccharification.35 In the koji samples with DOM 5 substrate, enhanced β-
303
glucosidase activities in rice koji were mainly correlated with a decrease in the flavonoid 14
ACS Paragon Plus Environment
Page 14 of 33
Page 15 of 33
Journal of Agricultural and Food Chemistry
304
glycosides and tricin rutinoside with corresponding increase in the relative levels of sugars
305
and flavonoid aglycone, tricin. The changes observed in the metabolite levels for DOM 0 koji
306
samples might have been impeded by the rice bran wall impervious to enzymatic hydrolysis.
307
Hence, following the rice bran hydrolysis, the relative levels of most metabolites was altered
308
in DOM 0 koji samples. The delay in metabolite release in case of brown rice koji
309
fermentation was reportedly linked to the impenetrable brine layer, which limits enzymatic
310
penetration and subsequent hydrolysis.10 Earlier, Yu et al. have reported that the protease and
311
glucoamylase activities for rice koji made using a brown rice substrate with 50–70% DOM
312
reach maximum following the 5 days fermentation with A. oryzae.36
313
The ABTS and FRAP assay determined antioxidant activities were considerably higher
314
in rice koji using substrates with DOM 0, 5, and 7, in comparison to those for DOM 9 and 11
315
koji samples, during the courses of fermentation. Hence, increasing the substrate's DOM
316
allows the enzymatic penetration through impervious rice bran and endosperm, hence
317
releasing the polyphenols, flavonoids, and free amino acids affecting the antioxidant
318
activities.13,14 Antioxidant activity prevents or limits oxidation, which is considered beneficial
319
for improving food quality and health. Evaluating antioxidant compounds from different
320
DOM of rice substrates provides useful information for commercial koji making owing to the
321
nutritional and functional properties of antioxidant metabolites.
322
Previously, it has been shown that flavonoid and phenol compounds derived from plants
323
possess high antioxidant activities.37 Pérez et al. have reported that the amino acid
324
composition of honey affects its free radical scavenging capacity.38 A known antioxidant
325
phytochemical i.e., ferulic acid is commonly found in rice bran, which reportedly alleviates
326
oxidative stress in various organic systems.39 We observed that the 96 h Rice koji with DOM
327
0 substrate contained the highest levels of antioxidant compounds. We conjecture that 15
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
328
antioxidant potentials of rice koji are mainly influenced by its flavonoids and phenolic acid
329
components, which synergistically constitutes to its various functional properties.
330
In conclusion, we observed that A. oryzae exhibited a unique overall metabolism
331
maneuvering the secretion of hydrolytic enzymes (amylase, β-glucosidase, and proteases)
332
ergo the metabolomes in rice koji fermented using the substrates with varying DOM.
333
Typically at the initial and final stages of koji fermentation, a variety of functional
334
metabolites were relatively higher in koji made with substrates having DOM 0, in comparison
335
to koji types made with substrates subjected to varying DOM. However, in the middle stages
336
of koji fermentation, the substrates with DOM 5–7 showed relatively higher contents of
337
metabolites intermediates of carbohydrate metabolism viz., sugars and sugar alcohols, organic
338
acids, phenolic acids, as well as lipid metabolism intermediates such as fatty acids and
339
lysophospholipids. The enhanced release of free metabolites in the koji samples was
340
influenced by the relatively higher amylase, β-glucosidase, and protease levels in rice
341
substrates with DOM 5-7 as compared to DOM 0. The present findings provide useful
342
insights for large-scale commercial production of rice koji that relies heavily on the DOM to
343
rationalize its raw substrate processing.
16
ACS Paragon Plus Environment
Page 16 of 33
Page 17 of 33
Journal of Agricultural and Food Chemistry
344
AUTHOR INFORMATION
345
Corresponding Author
346
*Phone: +82-2-2049-6177; fax: +82-2-455-4291; E-mail:
[email protected] 347
Notes
348
The authors declare no competing financial interest.
349 350
SUPPORTING INFORMATION
351
The list of significantly distinct metabolites from rice koji with different DOM during
352
fermentation identified by GC-TOF-MS (Table S1), list of significantly distinct metabolites
353
from rice koji with different DOM during fermentation identified by UHPLC-LTQ-ESI-IT-
354
MS/MS (Table S2), partial least square discriminant analysis (PLS-DA) score plot derived
355
from the GC-TOF-MS (A) and UHPLC-LTQ-ESI-IT-MS/MS data set of rice koji cultured
356
with A. oryzae according to the degree of milling (DOM) (Figure S1).
357 358
REFERENCES
359
1. Ray, R.C.; Montet, D. Microorganisms and fermentation of traditional foods. New York:
360
CRC Press, (Chapter 1), 2014.
361
2. Shin, D.; Jeong, D. Korean traditional fermented soybean products: Jang. J. Ethn. Foods
362
2015, 2, 2-7.
363
3. Bavaresco, L.; Vezzulli, S.; Civardi, S.; Gatti, M.; Battilani, P.; Pietri, A.; Ferrari, F. Effect
364
of Lime-Induced Leaf Chlorosis on Ochratoxin A, trans-Resveratrol, and ε-Viniferin
365
Production in Grapevine (Vitis vinifera L.) Berries Infected by Aspergillus carbonarius. J.
366
Agric. Food Chem. 2008, 56, 2085-2089. 17
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
367
4. Andújar-Ortiz, I.; Pozo-Bayón, M. A.; García-Ruiz, A.; Moreno-Arribas, M. V. Role of
368
specific components from commercial inactive dry yeast winemaking preparations on the
369
growth of wine lactic acid bacteria. J. Agric. Food Chem. 2010, 58, 8392-8399.
370
5. Chancharoongpong, C.; Hsieh, P.C.; Sheu, S.C. Enzyme production and growth of
371
Aspergillus oryzae S. on soybean koji fermentation. APCBEE Procedia 2012, 2, 57-61.
372
6. Bechman, A.; Phillips, R.D.; Chen, J. Changes in selected physical property and enzyme
373
activity of rice and barley koji during fermentation and storage. J. Food Sci. 2012, 77, 318-
374
322.
375
7. Zhu, Y.; Tramper, J. Koji-where East meets West in fermentation. Biotechnol. Adv. 2013,
376
31, 1448-1457.
377
8. Lee, D.E.; Lee, S.; Jang, E.S.; Shin, H.W.; Moon, B.S.; Lee, C.H. Metabolomic profiles of
378
Aspergillus oryzae and Bacillus amyloliquefaciens during rice koji fermentation. Molecules,
379
2016, 21, 773.
380
9. Kim, A.J.; Choi, J.N.; Kim, J.; Kim, H.Y.; Park, S.B.; Yeo, S.H.; Choi, J.H.; Liu, K.H.; Lee,
381
C.H. Metabolite profiling and bioactivity of rice koji fermented by Aspergillus strains. J.
382
Microbiol. Biotechnol. 2012, 22, 100-106.
383
10. Lee, D.E.; Lee. S.; Singh, D.; Jang, E.S.; Shin, H.W.; Moon, B.S.; Lee, C.H. Time-
384
resolved comparative metabolomes for koji fermentation with brown-, white-, and giant
385
embryo-rice. Food Chem. 2017a, 231, 258-266.
386
11. Yoshizaki, Y.; Yamato, H.; Takamine, K.; Tamaki, H.; Ito, K.; Sameshima, Y. Analysis of
387
volatile compounds in shochu koji, sake koji, and steamed rice by gas chromatography-mass
388
spectrometry. J. Inst. Brew. 2010, 116, 49-55. 18
ACS Paragon Plus Environment
Page 18 of 33
Page 19 of 33
Journal of Agricultural and Food Chemistry
389
12. Butsat, S.; Siriamornpun, S. Antioxidant capacities and phenolic compounds of the husk,
390
bran and endosperm of Thai rice. Food Chem. 2010, 119, 606-613.
391
13. Liu, L.; Guo, J.; Zhang, R.; Wei, Z.; Deng, Y.; Guo, J.; Zhang, M. Effect of degree of
392
milling on phenolic profiles and cellular antioxidant activity of whole brown rice. Food Chem.
393
2015, 185, 318-325.
394
14. Liu, K.I.; Zheng, J.B.; Chen, F.S. Relationships between degree of milling and loss of
395
Vitamin B, minerals, and change in amino acid composition of brown rice. LWT-Food Sci.
396
Technol. 2017, 82, 429-436.
397
15. Lee, S.; Lee, S.; Singh, D.; Oh, J.Y.; Jeon, E.J.; Ryu, H.S.; Lee, D.W.; Kim, B.S.; Lee,
398
C.H. Comparative evaluation of microbial diversity and metabolite profiles in doenjang, a
399
fermented soybean paste, during the two different industrial manufacturing processes. Food
400
Chem. 2017b, 221, 1578-1586.
401
16. Jang, Y.K.; Shin, G.R.; Jung, E.S.; Lee, S.; Lee, S.; Singh, D.; Jang, E.S.; Shin, D.J.; Kim,
402
H.J.; Shin, H.W.; Moon, B.S.; Lee, C.H. Process specific differential metabolomes for
403
industrial gochujang types (pepper paste) manufactured using white rice, brown rice, and
404
wheat. Food Chem. 2017, 234, 416-242.
405
17. Re, R.; Pellegrini, N.; Proteggente, A.; Pannala, A.; Yang, M.; Rice-Evans, C. Antioxidant
406
activity applying an improved ABTS radical cation decolorization assay. Free Radic. Biol.
407
Med. 1999, 26, 1231-1237.
408
18. Byeon, J.Y.; Choi, E.J.; Kim, W.J. Effect of low frequency (20-35 kHz) airborne
409
ultrasonication on microbiological and physicochemical properties of soybean koji. Food Sci.
410
Biotechnol. 2015, 24, 1035-1040. 19
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
411
19. Karthikeyan, A.; Sivakumar, N. Citric acid production by koji fermentation using banana
412
peel as a novel substrate. Bioresour. Technol. 2010, 101, 5552-5556.
413
20. Yin, X.; Li, J.; Shin, H.D.; Du, G.; Liu, L.; Chen, J. Metabolic engineering in the
414
biotechnological production of organic acids in the tricarboxylic acid cycle of
415
microorganisms: Advances and prospects. Biotechnol. Adv. 2015, 33, 830-841.
416
21. Kim, A.J.; Choi, J.N.; Kim, J.; Park, S.B.; Yeo, S.H.; Choi, J.H.; Lee, C.H. GC-MS based
417
metabolite profiling of rice koji fermentation by various fungi. Biosci. Biotechnol. Biochem.
418
2010, 74, 2267-2272.
419
22. Godswill, A.C. Sugar alcohols: chemistry, production, health concerns and nutritional
420
importance of mannitol, sorbitol, xylitol, and erythritol. Int. J. Adv. Res. 2017, 3, 31-66.
421
23. Blieva, R.K.; Safuani, Z.E.; Iskakbaeva, Z.A. Effect of various sources of nitrogen and
422
carbon on the biosynthesis of proteolytic enzymes in a culture of Aspergillus awamori 21/96.
423
Appl. Biochem. Microbiol. 2003, 39, 188-191.
424
24. Yousaf, M.; Irfan, M.; ulla Khokhar, Z.; Syed, Q.U.A.; Baig, S.; Iqbal, A. Enhanced
425
production of protease by mutagenized strain of Aspergillus oryzae in solid substrate
426
fermentation of rice bran. Sci. Int. 2010, 22, 119-123.
427
25. Kim, B.M.; Park, J.H.; Kim, D.S.; Kim, Y.M.; Jun, J.Y.; Jeong, I.H.; Nam, S.Y.; Chi, Y.M.
428
Effects of rice koji inoculated with Aspergillus luchuensis on the biochemical and sensory
429
properties of a sailfin sandfish (Arctoscopus japonicas) fish sauce. Int. J. Food Sci. Technol.
430
2016, 51, 1888-1899.
431
26. Sasse, A.; Hamer, S.N.; Amich, J.; Binder, J.; krappmann, S. Mutant characterization and
432
in vivo conditional repression identify aromatic amino acid biosynthesis to be essential for 20
ACS Paragon Plus Environment
Page 20 of 33
Page 21 of 33
Journal of Agricultural and Food Chemistry
433
Aspergillus fumigatus virulence. Virulence, 2016, 7, 56-62.
434
27. Shimizu, M.; Fujii, T.; Masuo, S.; Takaya, N. Mechanism of De Novo branched-chain
435
amino acid synthesis as an alternative electron sink in hypoxic Aspergillus nidulans cells.
436
Appl. Environ. Microbiol. 2010, 76, 1507-1515.
437
28. Kadir, S.A.; Wan-Mohtar, W.A.A.Q.I.; Mohammad, R.; Lim, S.A.H.; Mohammed, A.S.;
438
Saari, N. Evaluation of commercial soy sauce koji strains of Aspergillus oryzae for γ–
439
aminobutryic acid (GABA) production. J. Ind. Microbiol. Biotechnol. 2016, 43, 1387-1395.
440
29. Hayakawa, K.; Kimura, M.; Kasaha, K.; Matsumoto, K.; Sansawa, H.; Yamori, Y. Effect
441
of a γ-aminobutyric acid enriched dairy product on the blood pressure of spontaneously
442
hypertensive and normotensive Wistar-Kyoto rats. Brit. J. Nutr. 2004, 92, 411-417.
443
30. Yamatsu, A.; Yamashita, Y.; Maru, I.; Yang, J.; Tatsuzaki, J.; Kim, M. The improvement
444
of sleep by oral intake of GABA and Apocynum veneetum leaf extract. J. Nutr. Sci. Vitaminol.
445
2015, 61, 182-187.
446
31. Jakobs, C.; Jaeken, J.; Gibson, K.M. Inherited disorders of GABA metabolism. J. Inher.
447
Metab. Dis. 1993, 16, 704-715.
448
32. Oliveira, M.S.; Feddern, V.; Kupski, L.; Cipolatti, E.P.; Badiale-Furlong, E.; Souza-
449
Soares, L.A. Changes in lipid, fatty acids and phospholipids composition of whole rice bran
450
after solid-state fungal fermentation. Bioresour. Technol. 2011, 102, 8335-8338.
451
33. Abu, O.A.; Tewe, O.O.; Losel, D.M.; Onifade, A.A. Changes in lipid, fatty acids and
452
protein composition of sweet potato (Ipomoea batatas) after solid-state fungal fermentation.
453
Bioresour. Technol. 2000, 72, 189-192. 21
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
454
34. Friedman, M. Rice brans, rice bran oils, and rice hulls: composition, food and industrial
455
uses, and bioactivities in humans, animals, and cells. J. Agric. Food Chem. 2013, 61, 10626-
456
10641.
457
35. Rani, V.; Mohanram, S.; Tiwari, R.; Nain, L.; Arora, A. Beta-glucosidase: Key enzyme in
458
determining efficiency of cellulose and biomass hydrolysis. J. Bioprocess Biotech. 2014, 5, 1-
459
8.
460
36. Yu, K.W.; Lee, S.E.; Choi, H.S.; Suh, H.J.; Ra, K.S.; Choi, J.W.; Hwang, J.H.
461
Optimization for rice koji preparation using Aspergillus oryzae CJCM-4 isolated from a
462
korean traditional meju. Food Sci. Biotechnol. 2012, 21, 129-135.
463
37. Kong, S.; Lee, J. Antioxidants in milling fractions of black rice cultivars. Food Chem.
464
2010, 120, 278-281
465
38. Pérez, R.A.; Iglesias, M.T.; Pueyo, E.; González, M.; de Lorenzo, C. Amino acid
466
composition and antioxidant capacity of Spanish honeys. J. Agric. Food Chem. 2007, 55,
467
360-365.
468
39. Srinivasan, M.; Sudheer, A.R.; Menon, V.P. Ferulic acid: Therapeutic potential through its
469
antioxidant property. J. Clin. Biochem. Nutr. 2017, 40, 92-100.
470 471
FUNDING SOURCES
472
This work was supported by the National Research Foundation of Korea(NRF) grant funded
473
by the Korea government(MSIP) (No. NRF-2017M3C1B5019303) and funded by the
474
Strategic Initiative for Microbiomes in Agriculture and Food, Ministry of Agriculture, Food
475
and Rural Affairs, Republic of Korea (as part of the (multi-ministerial) Genome Technology 22
ACS Paragon Plus Environment
Page 22 of 33
Page 23 of 33
476
Journal of Agricultural and Food Chemistry
to Business Translation Program) (grant number 916005-2).
23
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
477
FIGURE CAPTIONS
478
Figure 1. Principal component analysis (PCA) score plot from the GC-TOF-MS (A) and
479
UHPLC-LTQ-IT-MS/MS (B) data set of rice koji cultured with A. oryzae according to the
480
degree of milling (DOM) ) (◆, ◆, 0; ◆, 5; ◆, 7; ◆, 9; ◆, 11 ; +, 0 hr; ✱, 12 h; ▲, 24 h; ■,
481
36 h; ▼, 48 h; ●, 72 h; ◆, 96 h).
482 483
Figure 2. Pathway for the relative levels of discriminant metabolites at different times of rice
484
koji with DOM as determined using the PLS-DA datasets (variable importance in projection
485
> 1.0, p-value < 0.1) for GC-TOF-MS analyses. The discriminant metabolites were further
486
correlated with corresponding steps in the biosynthetic pathways adapted from KEGG
487
database. The values represent the fold-change with respect to unfermented rice.
488 489
Figure 3. Changes in amylase (A), protease (B), β-glucosidase (C), ABTS (D), and FRAP (E)
490
of rice koji cultured with A. oryzae according to DOM during fermentation (■, 0; ■, 5; ■, 7;
491
■, 9; ■, 11).
492 493
Figure 4. Total mold count (A), moisture contents (B), pH (C), titratable acidity (D), total
494
sugar content (E), and total amino acid content (F) of koji cultured with A. oryzae according
495
to the degree of milling (DOM) during fermentation (■, 0; ■, 5; ■, 7; ■, 9; ■, 11).
24
ACS Paragon Plus Environment
Page 24 of 33
Page 25 of 33
Journal of Agricultural and Food Chemistry
496 497
Figure 1.
498
25
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
499 500
Figure 2.
501
26
ACS Paragon Plus Environment
Page 26 of 33
Page 27 of 33
Journal of Agricultural and Food Chemistry
502 503
Figure 3.
504
27
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
505 506
Figure 4.
507
28
ACS Paragon Plus Environment
Page 28 of 33
Page 29 of 33
508
Journal of Agricultural and Food Chemistry
Table 1. Information for degree of milling of rice .
Degree of milling
Proportion of the embryo bud and
(DOM)
bran layer in rice (%)
0
100
5
50
7
30
9
15
11
5
29
ACS Paragon Plus Environment
509
Journal of Agricultural and Food Chemistry
Page 30 of 33
510
Table 2. Column representations for the highest levels of discriminant metabolites in 5 rice
511
koji according to the DOM. Each column represents DOM color having metabolite of the
512
highest content at each time. (■, 0; ■, 5; ■, 7; ■, 9; ■, 11)
Fermentation time Metabolites
0h 7
12 h
0
5
9
11
0
alanine
'
'
'
'
'
asparagine
'
'
'
'
aspartic acid
'
'
'
γ-Aminobutyric acid
'
'
glutamic acid
'
glycine
24 h
5
7
9
11
0
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
Isoleucine
'
'
'
'
leucine
'
'
'
lysine
'
'
methionine
'
orinithine
36 h
5
7
9
11
0
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
phenylalanine
'
'
'
'
'
'
proline
'
'
'
'
'
pyroglutamic acid
'
'
'
'
serine
'
'
'
threonine
'
'
tryptophan
'
valine
48 h
5
7
9
11
0
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
myristic acid
'
'
elaidic acid
'
linoleic acid
72 h
5
7
9
11
0
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
linolenic acid
'
'
'
'
oleic acid
'
'
'
palmitic acid
'
'
stearic acid
'
'
96 h
5
7
9
11
0
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
5
7
9
11
'
'
' '
'
'
'
' '
'
'
'
'
' '
'
'
'
'
'
' '
'
'
'
'
'
'
' '
'
'
'
'
'
'
'
' '
'
'
'
'
'
'
'
'
' '
'
'
'
'
'
'
'
'
'
' '
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
' '
'
'
'
'
'
'
'
'
'
'
'
'
' '
'
'
'
'
'
'
'
'
'
'
'
'
'
' '
'
'
'
'
'
'
'
'
'
'
'
'
'
'
' '
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
' '
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
' '
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
' '
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
' '
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
' '
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
' '
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
' '
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
' '
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
' '
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
' '
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
' '
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
' '
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
' '
Amino acids
Fatty acids
Organic acids lactic acid
30
ACS Paragon Plus Environment
Page 31 of 33
Journal of Agricultural and Food Chemistry
4-hydroxybenzoic acid
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
' '
acetic acid
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
' '
citric acid
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
' '
ethanolamine
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
' '
fumaric acid
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
' '
malic acid
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
' '
salicylic acid
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
' '
shikimic acid
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
' '
succinic acid
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
' '
ferulic acid
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
' '
glycerol
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
' '
adonitol
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
' '
dulcitol
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
' '
gluconic acid
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
' '
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
Sugar & sugar alcohols
glucose glyceric acid
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
' '
glyceryl-glycoside
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
' '
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
' '
maltose Erythritol
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
' '
myo-inositol
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
' '
N-acetyl-D-glucosamine
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
' '
raffinose
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
' '
sorbitol
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
' '
sucrose
'
'
'
'
'
'
'
'
' '
xylose
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
' '
adenine
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
' '
guanine
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
' '
olemide
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
' '
uridine
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
' '
Nucleosides
Secondary metabolites apigenin-6-C-glucosyl-8-C-arabinoside isovitexin-2''-O-glucoside tricin-7-O-rutinoside tricin pinellic acid lysoPE14:0
31
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
lysoPC18:3 lysoPC14:0 lysoPE18:2 lysoPC16:1 lysoPC18:2 lysoPE16:0 lysoPC18:1
513 514
32
ACS Paragon Plus Environment
Page 32 of 33
Page 33 of 33
515
Journal of Agricultural and Food Chemistry
GRAPHIC FOR TABLE OF CONTENTS
516 517 518 519
33
ACS Paragon Plus Environment